Hepatitis B virus (HBV) is a hepatotropic virus that replicates by reverse transcription. It chronically infects ~350 million people and kills >600,000 annually. Therapy primarily employs nucleos (t)ide analogs that drive HBV to below the limit of detection in most patients. However, viral replication is not eradicated and viral replication resurges if drug is withdrawn, so therapy is life-long. Nevertheless, current treatment does clear HBV in some patients, so more patients could be cured by suppressing HBV further. Greater suppression of HBV will require new drugs that will probably be used in combination with the nucleos (t)ide analogs. HBV reverse transcription requires a viral DNA polymerase to synthesize new DNA and a viral ribonuclease H (RNaseH) to destroy the RNA after its conversion into DNA. Blocking either activity prevents HBV replication. Drugs against the RNaseH do not exist due to an inability to produce enzyme suitable for screening. We recently produced HBV RNaseH suitable for drug discovery. We screened 54 troponoid compounds, focusing on the ?-hydroxytropolones (?HTs), based on their ability to inhibit the HIV RNaseH and ?-thujaplicinol's inhibition of the HBV RNaseH. Thirteen compounds inhibited the HBV RNaseH, and the best had an IC50 value of 2.3 M. six compounds suppressed HBV replication in culture via blocking viral RNaseH activity. The best compound had an EC50 of 0.34 M and a therapeutic index of 94. A preliminary structure-activity relationship (SAR) was derived that provides guidance for optimization by medicinal chemistry. Here, Dr. Tavis (virologist) and Drs. Murelli and Meyers (synthetic and medicinal chemists) will collaborate to begin development of the ?HTs into potent anti-HBV drugs. Aim 1. Produce optimal ?HT inhibitors of HBV replication. We will synthesize ~320 ?HTs based on our preliminary SAR. These compounds will be tested in an iterative fashion for anti-HBV RNaseH activity in biochemical, viral replication, and cytotoxicity assays. Aim 2. Optimize cytotoxicity, ADME, PK, and efficacy parameters in a mouse model of HBV replication. Absorption, distribution, metabolism, excretion (ADME), initial pharmacokinetics (PK), and toxicity will be measured to guide compound design. Efficacy will be characterized in a mouse model of HBV replication for the leading inhibitors. Aim 3. Evaluate the potential for use of ?HT compounds in combination with nucleos(t)ide analogs. We will assess efficacy of the optimized ?HT compounds against common nucleos(t)ide analog- resistant HBV strains. We will also evaluate whether ?HT inhibitors work synergistically with the nucleos(t)ide analogs as predicted from their binding to physically distinct active sites on the HBV reverse transcriptase. This project will produce the first ?HT inhibitors designed against the HBV RNaseH and will define their potential to contribute to anti-HBV therapy. It may also produce lead compounds for drug development.